Abstract

Urothelial cancer (UC) is a well-known multifocal disease with frequent recurrences. The clonal origin of spatially separated UC foci is controversial. Here we propose to elucidate the multifocal UC genome by next generation sequencing (NGS) and thereby identify novel clonal heterogeneity among the lesions of a single bladder. Until now, all experiments aimed at understanding the molecular heterogeneity were generated mostly by candidate gene approach and older technologies. There is a lack of comprehensive molecular information in these multifocal lesions partly due to inadequate sample size for comprehensive molecular studies. Identification of mutational status at the gene level in different multifocal lesions will allow us to identify markers for prognostic classification, and predictive classification of response to UC therapies, as well as identify potential therapeutic targets.

To understand the molecular heterogeneity among the lesions of a single bladder, we used The Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) (ThermoFisher). The Ion AmpliSeq™ CCP was designed to target all exons of 409 key tumor suppressor genes (TSGs) and oncogenes most frequently cited and mutated in cancer. We tested 41 lesions from 16 cystectomized bladders. Among these lesions, we performed an initial analysis of a total of 21 lesions and 8 germ line controls from 8 patients. We further technically validated selected mutational events found by NGS by a complementary approach, namely droplet digital PCR (ddPCR). We then compared the intra- and inter-tumoral mutation profiles. Two separate lesions were used for analysis in 3 patients and 3 lesions were taken from each of the remaining 5 patients. Our initial analysis showed mutational heterogeneity among the lesions of most of the samples. Briefly, two of the patients had one clone found at all sites, while the rest of the patients showed more variation amongst their lesions. For instance, ARID2, TRRAP, PDGFB, and FBXW7 were mutated in two out of three lesions in one of the patients, indicating hotspot for heterogeneous mutational events. Three mutational events were confirmed by ddPCR, denoting the accuracy of our NGS data analysis.

Our analysis demonstrates that targeted next generation sequencing is a sensitive and reliable method for interrogating the molecular landscape of bladder cancer, and indicates that both clonal and spontaneous events can account for multifocal bladder cancer.

Our data suggests that sequencing of multiple lesions from an individual patient is necessary to accurately elucidate the mutation profile and tailor appropriate targeted treatment. Further analysis in an extended number of samples is necessary to fully understand the molecular heterogeneity at the mutational level in urothelial cancer.